Abstract: Spodoptera ornithogalli (Guenée) (Lepidoptera) is a phytophagous insect of familyNoctuidae. In Colombia, this specie had been reported in several crops including cotton andornamental flowers with economic importance. The Nucleopolyhedrovirus of Spodopteraornithogalli (SoNPV) is a natural enemy for the larvae stage. In this work, S. ornithogalli larvaecollected from citric crop were reared until dead by viral symptoms. The SoNPV obtained weresubject to morphological and biological characterization. Additionally, restriction endonucleaseanalysis (REN) patterns were made using four restriction endonucleases. The size of thepolyhedra showed high variation although the most frequent range size was inferior compared toS. frugiperda polyhedra. Mean lethal concentration (LC50) determined in neonates larvae was1x105 OBs/mL and the yield per unit weight of the larvae was found to be 2×109 OBs/g. TheREN showed a differential pattern compared with others baculoviruses. This is a preliminarystudy of SoNPV native from Colombia and could be the base for future works in order to developa biopesticide.